Quantification of human immunodeficiency virus type 1 RNA levels in plasma by using small-volume-format branched-DNA assays

We have developed small-volume (50 or 250 microl)-format branched-DNA assays for human immunodeficiency virus type 1 (HIV-1) RNA for use with specimens in which the volume is limited and/or a high viral load is anticipated. These formats exhibited good correlation with the standard 1-ml format; high specificity, reproducibility, and linearity; and no significant difference in the quantification of HIV-1 subtypes.     

Evaluation of human immunodeficiency virus (HIV) type 1 RNA levels in cerebrospinal fluid and viral resistance to zidovudine in children with HIV encephalopathy

The amount of human immunodeficiency virus (HIV) type 1 RNA and the presence of a codon 215 mutation indicative of zidovudine resistance were evaluated in cerebrospinal fluid (CSF) and plasma obtained from HIV-1-infected children. The level of HIV-1 RNA in CSF was highest in children with severe encephalopathy (n = 25; median, 430 copies/mL; range, 0-2.2 x 10(5) copies/mL) followed by the moderately encephalopathic (n = 7; median, 330; range, 0-1130) and nonencephalopathic groups (n = 9; median, 0; range, 0-566) (P = .007). There was no correlation between CSF and plasma HIV-1 RNA levels. Five of 7 children with the codon 215 mutation in CSF had a progression of encephalopathy, while all 8 children with wild type codon 215 had improved or stable disease during zidovudine treatment (P = .007). These findings suggest that increased viral replication and emergence of drug-resistant HIV-1 variants within the central nervous system may play a role in progression of HIV encephalopathy.     

Evaluation of the Nuclisens HIV-1 QT assay for quantitation of human immunodeficiency virus type 1 RNA levels in plasma

Nuclisens HIV-1 QT is a new version of the NASBA HIV-1 QT assay for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma. The specificity of this assay was 100% in one laboratory and 99%-with nonrepeatability of the initial false positive-in another. The test was linear between 2.0 and 6.0 log RNA copies per ml. According to the input HIV-1 RNA concentration, accuracy varied from -0.11 to +0.10 log RNA copy per ml and precision varied from 0.66 to 0.14 log RNA copy per ml. Reproducibility decreased when the HIV-1 RNA level was near the lower limit of quantitation of the test. HIV-1 RNA could be quantitated by Nuclisens HIV-1 QT in 36% (laboratory 1) and 24% (laboratory 2) of clinical samples with HIV-1 RNA levels lower than the lower limit of quantitation by NASBA HIV-1 QT. Nuclisens HIV-1 QT was not suitable for measurement of RNA from clade G and group O HIV-1 strains.     

Ultrasensitive reverse transcription-PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma

With the recent introduction of combination therapy, human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma have been dramatically reduced, frequently to below the limit of quantitation (400 copies/ml of plasma) of the AMPLICOR HIV-1 MONITOR Test (Roche Diagnostic Systems). To achieve enhanced sensitivity of the AMPLICOR HIV-1 MONITOR Test, a modified specimen preparation procedure that allows input of RNA from 10-fold more plasma per amplification reaction was developed. This "ultrasensitive" method allows the accurate quantitation of plasma HIV-1 RNA levels as low as 50 copies/ml. A precision study yielded average within-run and between-run coefficients of variation (CV) of 24.8 and 9.6%, respectively. A multicenter reproducibility study demonstrated that the laboratory-to-laboratory reproducibility of this assay is good, with an average CV of 32%. The linear range of this test is between 50 and 50,000 copies/ml of plasma. RNA concentrations measured by the ultrasensitive and standard HIV-1 MONITOR tests exhibited good agreement within the shared linear range of the two methods. The two measurements were within a factor of 2 for 91% of the specimens tested, with the concentration measured by the ultrasensitive method being only slightly lower (median, 22% lower). Preliminary studies suggest that this assay will prove to be useful for predicting the stability of viral suppression in patients whose RNA levels drop below 400 copies/ml in response to highly active antiretroviral therapy.     

Genotypic and phenotypic characterization of human immunodeficiency virus type 1 variants isolated from patients treated with the protease inhibitor nelfinavir

Nelfinavir mesylate (formerly AG1343) is a potent and selective inhibitor of human immunodeficiency virus (HIV) protease approved for the treatment of individuals infected with HIV. Nucleotide sequence analysis of protease genes from plasma HIV type 1 (HIV-1) RNA revealed a unique aspartic acid (D)-to-asparagine (N) substitution at residue 30 (D30N) in 25 of 55 patients treated with nelfinavir for a median of 13 weeks. Although the appearance of D30N was occasionally associated with concurrent or sequential emergence of other changes (e.g., at residues 35, 36, 46, 71, 77, and 88), genotypic changes associated with phenotypic resistance to other protease inhibitors were not observed (e.g., at residues 48, 50, 82, and 84) or were only rarely observed (e.g., at residue 90). In phenotypic assays, viral isolates with high-level resistance to nelfinavir remained susceptible to indinavir, saquinavir, ritonavir, and amprenavir (formerly VX-478/141W94). Similar results were observed in phenotypic assays utilizing HIV-1 NL4-3, which contained the D30N substitution alone or in combination with substitutions at other residues (e.g., residues 46, 71, and 88). These data indicate that the initial pathway of resistance to nelfinavir is unique and suggest that individuals failing short courses of nelfinavir-containing regimens may respond to regimens containing other protease inhibitors.     

Serum levels of human immunodeficiency virus type 1 (HIV-1) RNA after seroconversion: a predictor of long-term mortality in HIV infection

A cohort of 79 homosexual men with documented dates of human immunodeficiency virus type 1 (HIV-1) seroconversion and baseline CD4 cell counts of > or = 500/microL were followed for up to 11.5 years. HIV-1 RNA was measured from stored sera obtained a median of 7 months after the estimated seroconversion date. AIDS progression and mortality among the men were studied, stratified by median baseline levels of HIV-1 RNA. AIDS progression rates at 11.5 years were 69% and 34%, respectively, among those with higher versus lower than median baseline virus loads (> or = 3040 copies/mL; P = .002), and mortality rates were 61% and 27%, respectively (P = .003). Survival curves continued to diverge throughout the 11.5 years, suggesting that the future clinical course of HIV-1 infection may already be determined at the earliest phases of disease. Initiation of definitive treatment very early in HIV-1 infection may be essential.     

Effects of specimen collection, processing, and storage conditions on stability of human immunodeficiency virus type 1 RNA levels in plasma

To define the optimal blood collection parameters for plasma human immunodeficiency virus type 1 (HIV-1) viral load testing, plasma HIV-1 RNA levels were quantitated with the NASBA HIV-1 RNA QT System from blood specimens that were collected, processed, and stored under a variety of conditions that might have affected HIV-1 RNA stability. We determined that when whole blood was processed within 2 h of specimen collection the levels of HIV-1 RNA detected in EDTA-, heparin-, and acid citrate dextrose (ACD)-anticoagulated plasma samples were comparable. The levels of HIV-1 RNA in serum specimens (mean = 4.126 log units) were significantly lower (P < 0.01) than the levels in corresponding plasma samples (mean = 4.501 log units). One cycle of freeze-thaw (-70 degrees C) did not significantly reduce the level of HIV-1 RNA detected in EDTA-, heparin-, or ACD-anticoagulated plasmas. The EDTA-anticoagulated plasmas showed the smallest decrease in HIV-1 RNA copies (0.050 log units). HIV-1 RNA levels decreased over a 6-month time period in serum as well as in EDTA-, ACD-, and heparin-anticoagulated plasmas stored at -70 degrees C. However, the only significant decreases were for serum (mean decrease = 0.317 log units) and heparin-anticoagulated samples (mean decrease = 0.384 log units). A comparison of the levels of HIV-1 RNA in cell-free plasma collected in VACUTAINER EDTA Plasma Preparation Tubes and in standard VACUTAINER EDTA tubes determined that HIV-1 RNA levels were stable for up to 30 h after collection when stored at either room temperature (mean standard deviation [SD] = +/- 0.101 log units) or at 4 degrees C (mean SD = +/- 0.102 log units) as cell-free plasma or as EDTA-anticoagulated whole blood (mean SD = +/- 0.109 log units). These data indicate that EDTA-anticoagulated plasma is the most suitable and stable matrix for HIV-1 RNA quantitation.     

Intra-assay performance characteristics of five assays for quantification of human immunodeficiency virus type 1 RNA in plasma

Three kits (Roche AMPLICOR human immunodeficiency virus type 1 [HIV-1] Monitor, Chiron enhanced-sensitivity bDNA, and Organon Teknika NASBA HIV-1 QT) and two in-house assays (from National Genetics Institute and Baylor College of Medicine) were compared with a blinded panel. The results were evaluated as to intra-assay sensitivity, precision, and ability to detect differences in a dilution series.     

Dynamics of viral replication in infants with vertically acquired human immunodeficiency virus type 1 infection

About one-third of vertically HIV-1 infected infants develop AIDS within the first months of life; the remainder show slower disease progression. We investigated the relationship between the pattern of HIV-1 replication early in life and disease outcome in eleven infected infants sequentially studied from birth. Viral load in cells and plasma was measured by highly sensitive competitive PCR-based methods. Although all infants showed an increase in the indices of viral replication within their first weeks of life, three distinct patterns emerged: (a) a rapid increase in plasma viral RNA and cell-associated proviral DNA during the first 4-6 wk, reaching high steady state levels (> 1,000 HIV-1 copies/10(5) PBMC and > 1,000,000 RNA copies/ml plasma) within 2-3 mo of age; (b) a similar initial rapid increase in viral load, followed by a 2.5-50-fold decline in viral levels; (c) a significantly lower (> 10-fold) viral increase during the first 4-6 wk of age. All infants displaying the first pattern developed early AIDS, while infants with slower clinical progression exhibited the second or third pattern. These findings demonstrate that the pattern of viral replication and clearance in the first 2-3 mo of life is strictly correlated with, and predictive of disease evolution in vertically infected infants.     

Genotypic characterization of human immunodeficiency virus type 1 in Greece. Multicentre Study on HIV-1 Heterogeneity

The HIV-1 subtype distribution in 83 HIV-1-seropositive individuals living in Greece was investigated by using the heteroduplex mobility assay (HMA), DNA sequencing, and phylogenetic analysis. The results revealed that partial HIV-1 gp120 sequences from 71 (86%) patients were subtype B, 5 (6%) were subtype A, 4 were subtype D (5%), 2 (2%) were subtype C, and 1 (1%) was subtype I. The subtype I isolate was documented in an intravenous drug user. A high prevalence (90-100%) of B isolates among intravenous drug users, hemophiliacs, and homosexual men was observed, in contrast to heterosexuals, among whom non-B subtypes seemed to be common (42.9%, p < 0.001). Among the Greek population subtype B is the most frequent (94%), in contrast to the high prevalence (57%) of non-B isolates found in emigrants living in Greece (p < 0.001). A heterosexual transmission case of subtype D in a Greek individual not traveling abroad was also documented. The broad HIV-1 diversity in Greece may be explained by population movements, such as migration and traveling.     

Cervical human immunodeficiency virus type 1 shedding is associated with genital beta-chemokine secretion

Forty human immunodeficiency virus type 1 (HIV-1)-infected women participated in a cross-sectional study of possible correlations between chemokine receptor (CCR5 and/or CCR2B) genotype, HIV-1 RNA and DNA load, and beta-chemokine levels (RANTES, MIP-1alpha, MIP-1beta) in blood and cervix. HIV-1 nucleic acid and beta-chemokines were found in all patient blood samples and in more than half of the cervical samples regardless of CCR5 or CCR2B genotype. High beta-chemokine concentrations were in general associated with high virus loads in blood and cervix. In the blood, the proviral DNA load was significantly correlated with the MIP-1alpha concentration, whereas the DNA load in cervix was significantly associated with the MIP-1beta concentration. The cervical viral RNA load was significantly associated with levels of all three chemokines. Thus, when HIV-1 shedding was highest in the genital tract, it was associated with other combinations of beta-chemokines than virus load in blood, suggesting that local immune reactions strongly influence virus load in the cervical compartment.     

Control of human immunodeficiency virus type 1 RNA metabolism: role of splice sites and intron sequences in unspliced viral RNA subcellular distribution

In the course of examining the various factors which affect the metabolism of human immunodeficiency virus type 1 (HIV-1) RNA, we examined the role of intron sequences and splice sites in determining the subcellular distribution of the RNA. Using in situ hybridization, we demonstrated that in the absence of Rev, unspliced RNA generated with an HIV-1 env expression construct displayed discrete localization in the nucleus, coincident with the location of the gene and not associated with SC35-containing nuclear speckles. Expression of Rev resulted in a disperse signal for the unspliced RNA throughout both the nucleus and the cytoplasm. Subsequent fractionation of the nucleus revealed that the majority of unspliced viral RNA within the nucleus is associated with the nuclear matrix and that upon expression of Rev, a small proportion of the unspliced RNA is found within the nucleoplasm. Mutations which altered splice site utilization did not alter the sequestration of unspliced RNA into discrete nuclear regions. In contrast, a 2.2-kb deletion of intron sequence resulted in a shift from discrete regions within the nucleus to a disperse signal throughout the cell, indicating that intron sequences, and not just splice sites, are required for the observed nuclear sequestration of unspliced viral RNA.     

Increased plasma HIV type 1 RNA levels are associated with low levels of non-MHC class I-restricted cytotoxicity

2329 subjects (blood donors and patients) from various areas of the Sultanate of Oman were investigated for the presence of HTLV-I antibody by as enzyme immunoassay (EIA) method. 10 subjects (0.4%), including 9/1586 blood donors (0.6%) and 1/165 patients with sexually transmitted diseases (0.6%), were found to be EIA seropositive with a regional variation in seroprevalence of 0-14%. 6/9 EIA seropositive samples from blood donors yielded 'indeterminate' results on Western immunoblot analysis (WBA). A much larger survey with additional confirmatory assays such as a radioimmunoprecipitation assay (RIPA), should provide a more conclusive picture of the prevalence of this retroviral infection in the Sultanate.     

Recognition of prominent viral epitopes induced by immunization with human immunodeficiency virus type 1 regulatory genes

Mice immunized with the regulatory genes nef, rev, and tat from human immunodeficiency virus type 1 developed both humoral and cellular immune responses to the gene products Nef, Rev, and Tat. This study demonstrates that it is feasible to induce immune reactions to all of these regulatory gene products. Humoral responses were seen after DNA boosts, while potent T-cell proliferative responses were noted already after a single immunization. A Th1-directed immune response was demonstrated early after immunization. A 3- to 75-fold-stronger T-cell response was seen in animals receiving DNA epidermally compared to that in animals receiving intramuscular injections. Nef, Rev, and Tat putative B- and T-cell epitopes were clearly mapped by using peptides derived from the regulatory proteins and were similar to those which are detected in human immunodeficiency virus infection. Although immunization by the Nef, Rev, and Tat proteins raised high immunoglobulin G titers in serum, the epitope spreading appeared broader after DNA immunization. The combination of all of these regulatory genes together with two genes for structural proteins, the envelope and gag genes, demonstrated that a combined approach is feasible in that reactivities to all antigens persisted or were even augmented. No interference between plasmids was noted.     

Reproducibility and performance of the second-generation branched-DNA assay in routine quantification of human immunodeficiency virus type 1 RNA in plasma

We examined the reproducibility of a second-generation branched-DNA (bDNA) assay (Quantiplex HIV RNA 2.0) for quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma by retesting 325 specimens on separate runs and on different lots. The performance of the bDNA test was also assessed by data analysis obtained during routine testing of 15,365 specimens. Upon retesting, 96 and 86% of specimens displaying RNA levels above 5,000 and between 500 and 5, 000 copies/ml, respectively, showed less than a 0.3 log10 (twofold) difference with their initial values. Assay variability was found to increase as viral load decreased. Overall, the bDNA version 2.0 assay was found to be a reproducible and efficient test for routine quantification of HIV-1 RNA in plasma.     

Increased mortality associated with vitamin A deficiency during human immunodeficiency virus type 1 infection

OBJECTIVE: To determine whether plasma vitamin A levels are associated with immunologic status and clinical outcome during human immunodeficiency virus type 1 (HIV-1) infection. PATIENTS AND METHODS: Analysis of vitamin A levels, CD4 T cells, complete blood cell count, and serologic markers for liver disease in a random subsample of 179 subjects from a cohort of more than 2000 intravenous drug users with longitudinal follow-up to determine survival. RESULTS: Mean (+/- SE) follow-up time was 22.8 +/- 1.1 months, and 15 subjects died during follow-up. More than 15% of the HIV-1-seropositive individuals had plasma vitamin A levels less than 1.05 mumol/L, a level consistent with vitamin A deficiency. The HIV-1-seropositive individuals had lower mean plasma vitamin A levels than HIV-1-seronegative individuals (P < .001). Vitamin A deficiency was associated with lower CD4 levels among both seronegative individuals (P < .05) and seropositive individuals (P < .05). In the HIV-seropositive participants, vitamin A deficiency was associated with increased mortality (relative risk = 6.3; 95% confidence interval, 2.1 to 18.6). CONCLUSION: Vitamin A deficiency may be common during HIV-1 infection, and vitamin A deficiency is associated with decreased circulating CD4 T cells and increased mortality. Vitamin A is an essential micronutrient for normal immune function, and vitamin A deficiency seems to be an important risk factor for disease progression during HIV-1 infection.     

Depressive syndromes and symptoms in subjects with human immunodeficiency virus (HIV) infection

The association between the infection produced by the human immunodeficiency virus (HIV) and syndromal or subsyndromal depression has been the topic of several studies in recent years. The results of the WHO Neuropsychiatric AIDS Study, conducted in the five geographical areas predominantly affected by the HIV epidemic, suggest that the symptomatic stages of HIV infection are associated with an increased prevalence of depressive symptoms, and, at least in some contexts in which the spreading of the infection is more recent and the social rejection of HIV-seropositive subjects is harsher, may also be associated with an increased prevalence of a syndromal diagnosis of depression.